We propose to continue the investigation of the dynamics of ligand binding to heme proteins by flash photolysis. Heme proteins are ideal for such studies because the same active center, heme, performs very different functions, from oxygen storage to catalysis, in different proteins. The long range goal of our work is to understand how the protein primary sequence and tertiary structure control specificity and govern the catalytic and storage properties at the active center. In our present work, under grant GM 18051, we have demonstrated that measurements over wide ranges in temperature (2-350 K), time (10 to the minus 6th power) to (10 to the 3rd power sec), and pressure (0-2 kbar) are necessary to obtain insight into the processes at the active center. Low-temperature studies thus are essential for the elucidation of connections between structure and function. We have shown that access to the active site is governed by multiple barriers. Comparison between protoheme and heme proteins indicates that the innermost potential barrier is produced by the active center, the outermost by the solvent, and the intermediate ones by the protein. The activation enthalpy of the innermost barrier is not sharp, but distributed. The distribution is presumably caused by different conformational states that characterize the protein. Below 20 K, rebinding after photodissociation takes place by quantum-mechanical tunneling through the innermost barrier. To exploit the discoveries made so far, we propose to extend our measurements down to 10 to the minus 8th power sec, employ infrared spectroscopy, electron spin resonance, and Mossbauer effect, to observe the transient phenomena after photodissociation, continue the high-pressure work at low temperatures to determine all relevant activation volumes, look at chemically and genetically modified proteins and at model compounds, use ligands other than CO and O2, and explore the effects of solvents and specific compounds (pharmaceuticals) bound to allosteric sites.